Cancer is a serious threat to human life and health. According to data released by the World Health Organization, the new cases of cancers worldwide reached 14.1 million in 2012; cancer related deaths reached 8.2 million people. In 2008, these numbers were 1.27 million and 0.76 million respectively. In recent years, with the increase of aging population and environmental pollution, the occurrence and mortality rate of cancer have accelerated, and cancer has become one of the main causes of human death. It is expected that the world's new cases of cancer will reach 19.3 million people by 2025. Therefore, the new method of prevention and treatment for cancer is in urgent need.
According to the statistics, the top three cancers with the high occurrence rate were lung cancer (13%), breast cancer (11.9%) and colon cancer (9.7%), the top three cancers with the highest death rate were lung cancer (19.4%), liver cancer (9.1%) and gastric cancer (8.8%). Lung cancer has become the number one killer in cancer related death, in which non-small cell lung cancer (NSCLC) is the most common and accounts for 80% of the total number of lung cancers. Because of its lack of obvious early symptoms, most patients with lung cancer have developed into the middle or late stage when diagnosed, and thus missed the opportunity for early treatment Currently, the treatment for cancer mainly includes surgical treatment, radiotherapy and chemotherapy. The benefits with surgical treatment were obvious, but the recurrence and metastasis of tumor could happen easily. Radiation therapy can change the structure of the biological molecules. Thus, it can destroy the cancer cells. This method also has a strong side effect on normal cells. Chemotherapy uses chemical drugs to kill tumor cells, but it can also damage normal cells, causing obvious side effects.
In recent years, the tumor cell biology and genetics have been developed rapidly. The research on cancer gene, cell apoptosis and tumor angiogenesis has evolved to the level of molecular biology, the mechanism of tumor cells from molecular biology is being understood gradually, and the new ideas and methods of treatment are proposed continuously. Molecular targeted therapy provides a new way of cancer treatment. The study shows that the occurrence and development of tumor involve many signal transduction processes in living organisms. The molecular targeted drug is directed to malignant tumor tissues and cells based on specific biological targets. It has less toxic side effects and higher efficiency than the traditional chemotherapy does. As a result, cancer molecular targeted medicines have become the hot spot in the field of cancer research.
The molecular targeted therapy is a kind of therapy in which, on the basis of the molecular biology, the specific structure of the tumor tissue or cells is used as a target, and corresponding therapeutic medicines are designed to be able to combine with target molecules to achieve direct therapy or guiding therapy. This new method of cancer treatment is to reverse the malignant biological behavior of tumor cells at a molecular level so as to achieve the goal of inhibiting tumor growth. Unlike the conventional cytotoxic drugs, molecular targeted drugs can selectively kill the cancer cells by specifically acting on some specific sites (which normally are not expressed or less expressed in normal cells) so as to provide high safety, good tolerability, less toxic and side effect. For this reason, the molecular targeted therapy has a very big advantage and a broad application prospect.
As a targeted method of treatment, molecular targeted therapy needs to identify the biological targets in the first place. Currently, the common biological targets include oncogenes, anti-oncogenes and growth factor and its receptor, tumor angiogenesis factor, protein kinase and its signal transduction pathway, telomere and telomerase, DNA topoisomerase, histone deacetylase etc. There are multiple methods for testing the activity of the biological targets, including immunohistochemistry (IHC) to detect the protein expression, fluorescence in situ hybridization (FISH) or chromogenic in situ hybridization (CISH) to detect the number of gene copies, and polymerase chain reaction (PCR) to detect gene mutation. In many detection technologies, immunohistochemistry is the simplest, cheapest, and most commonly used.
Receptor tyrosine kinase (RTK) is the largest class of enzyme-linked receptors. It is both a receptor and an enzyme, which is capable of being bound by its ligand, allowing the phosphorylation of the target protein. All the RTKs are composed of three parts: the extracellular domain of the ligand binding site, the single transmembrane hydrophobic alpha helical region, and the intracellular domain involving receptor tyrosine kinase activity. At present, there are more than 50 RTKs, including epidermal growth factor receptor, platelet growth factor receptor, fibroblast growth factor receptor and vascular endothelial growth factor receptor
In the absence of binding ligand, RTKs exist in the form of monomer without any bioactivity. Once a ligand binds to the extracellular domain of the receptor, the two receptor monomers form a receptor dimer by the process of polymerization, which leads to the activation of the receptors and the phosphorylation of the tyrosine residues. The phosphorylation transforms the intracellular domain of the receptor into a signal complex which activates a series of biochemical reactions in the cells involving cell proliferation and survival or combines different signals to create a comprehensive cell response (such as cell proliferation). Thus, RTKs play a key role in cell signal transduction.
Research found that over-expression or over-activation of the receptor tyrosine kinase has been observed in many cancer cells, for example, the over-expression of epidermal growth factor receptor in epithelial tumor cells, the over-expression of platelet growth factor receptor in glioma. Over-expression of the tyrosine receptor activates the downstream signal transduction pathway, leading to the abnormal transformation and proliferation of the cells, and promotes the development of tumor.
Epidermal growth factor receptor (EGFR) is a class of receptor tyrosine kinase, which is the expression product of proto-oncogene c-erbB1 and belongs to the HER/ErbB family. The receptor family includes four members, i.e., HER1 (EGFR/erbB-1), HER2 (neu/erbB-2), HER3 (erbB-3) and HER4 (erbB-4). EGFR is widely distributed in the surface of mammalian epithelial cells, fibroblasts, glial cells, and other cells. The signal pathway plays an important role in regulating the physiological processes of cells.
From structure perspective, EGFR is a transmembrane protein, made of 1186 amino acids. It is divided into extracellular domain (ECD), transmembrane domain (TM), and intracellular domain (D). The intracellular structure contains one tyrosine kinase domain and multiple auto phosphorylation sites. After phosphorylation, these tyrosine residues bind specifically to the downstream protein of the signal transduction pathway, thereby activating the EGFR signaling pathway and completing the conduction and transfer process of the signaling from the extracellular cell to the intracellular cell. There are 6 known EGFR ligands, including epidermal growth factor (EGF), transforming growth factor α(TGFα), AmpHiregulin, Bctacelluin (BTC), Heparin-binding EGF (HBEGF), Epiregulin (EPR), and EGF and TGFα are the two most important ligands of EGFR. The binding of a ligand with a receptor results in an important conformational change, causing the dimerization of the receptors, which leads to the phosphorylation and stimulation of numerous intracellular signal transduction pathways involved in cell proliferation, apoptosis, migration, and survival.
Research shows that EGFR expresses in all the normal epidermal cells, but about ⅓ of human tumor has abnormal expression of EGFR, including head and neck squamous cell carcinoma (HNSCC), malignant glioma, non-small cell lung cancer (NSCLC), breast cancer, colon cancer. The possible mechanism is that the high expression of EGFR increases the downstream signal transduction; mutant EGFR receptor or ligand expression leads to the increase of sustained activation of EGFR; abnormal signal transduction pathway activation, etc.
Upon the external activation, EGFR forms a receptor dimer by the process of polymerization. The binding of ATP into EGFR receptor leads to the phosphorylation of intracellular tyrosine residues, which further activates the 3 main signal transduction pathways: (1) Ras2/Raf2/MAPK pathway, this pathway activation can catalyze the nuclear transcription factor of many serine/threonine phosphorylation, promote gene transcription, cell division and cell cycle; (2) the PI3K/Akt/mTOR pathway, which is an important anti-apoptotic pathway, and is associated with angiogenesis; (3) the JAK/STAT pathway whose activation can promote cell proliferation and prolong cell survival. These in turn trigger signaling pathway and control gene transcription, cell proliferation, differentiation and survival, ultimately mediated cell differentiation, survival, migration, invasion, adhesion and cell damage and repair process. Therefore, blocking the EGFR signaling pathway can inhibit the growth of tumor cells. As result, EGFR is an important target for cancer targeted therapy.
EGFR is overexpressed in many tumor cells, which leads to the uncontrolled growth of tumor cells and the increased degree of malignancy. At present, a series of anticancer drugs targeting EGFR have been developed, and some of them have been used in clinics. There are two types of drugs targeting EGFR: (1) monoclonal antibody binding to the extracellular domain of EGFR, such as cetuximab (Cetuximab, Erbitux, MCC225), Matuzumab (EMD72000) and ABX-EGF; (2) small molecule inhibitors binding to the intracellular kinase domain of tyrosine (tyrosine kinase inhibitor, TK1), such as gefitinib, erlotinib (Gefitinib/Iressa/ZD1839) and AG-1478 (Erlotinib/Tarceva/OSI-774).
The EGFR monoclonal antibody (cetuximab and panitumumab) is a human-mouse chimeric IgG monoclonal antibody aiming at EGFR extracellular domain. This EGFR monoclonal antibody has a strong affinity for EGFR. It has the function of blocking the binding site of the growth factor, preventing ligand-induced receptor from activation and phosphorylation, inhibiting tyrosine kinase from activation, blocking the signal transduction pathway relating to tumor cell proliferation, inhibiting cells from proliferation and promoting apoptosis. (DINF, MARTINI M, MOLINARI F, et al. Wild-type Braf is required for response to panitumumab or cetuximab in metastatic colorectal cancer, Journal of Clinical Oncology, 2008, 26(35): 5705-5712)
The small molecule tyrosine kinase inhibitor EGFR-TKI can competitively bind to the ATP binding site of EGFR and inhibit the phosphorylation of the receptor, thereby blocking the conduction of the downstream signal. The aniline quinazoline compounds show good inhibitory effect of the EGFR and the best selectivity for a class of tyrosine kinase inhibitors (Zhang Ke, Xie Guangru, Pan Zhanyu, small molecule tyrosine kinase inhibitors in the treatment of non-small cell lung cancer, clinical research progress, China, 2006, 33 (2):115-118; CIARDIELLO F, TORTORA G, EGFR antagonists in cancer treatment, New England Journal of Medicine, 2008, 358 (11): 1160-1174). Currently, some of the small molecule EGFR inhibitors on the market are based on the structure of aniline quinazoline.
(1) Gefitinib (Gefitinib/Iressa/ZD1839). Gefitinib, also known as gefitinib or Iressa, was developed by AstraZeneca as a selective and reversible EGFR tyrosine kinase inhibitors. It was approved in May 2003 by the US FDA for the treatment of advanced non-small cell lung cancer after chemotherapy failure. It was approved in China in March 2005. Gefitinib could competitive binding to the epidermal growth factor receptor tyrosine kinase (EGFR-TK) binding sites of catalytic region, blocking its downstream signaling, growth, metastasis and vascular and block tumor growth, apoptosis and induce tumor cells to anti-tumor effect (1, CIARDIELLO F, CAPUTO R, BIANCO R, et al. Inhibition of growth factor production and angiogenesis in human cancer cells by ZD1839 (Iressa), a selective epidermal growth factor receptor tyrosine kinase inhibitor. Clinical Cancer Research, 2001, 7:1459-1465; 2, BARKER A J, GIBSON K H, GRUNDY W, et al. Studies Leading to the Identification of ZD1839 (Iressa): An Orally Active, Selective Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Targeted to the Treatment of Cancer. Bioorganic & Chemistry Letters. 2001, 11:1911-1914).
Gefitinib is not only effective in the treatment of advanced non-small cell lung cancer, improving the symptoms relating to the disease, but also inhibit other solid tumors, including prostate cancer, breast cancer, head and neck cancer, gastric cancer, colon cancer. It can also improve the anti-tumor activity of chemotherapy, radiotherapy and hormone therapy.
(2) Erlotinib (Erlotinib/Tarceva/OSI-774). Erlotinib or Tarceva was approved in September 2002 by the FDA as the standard regimen for non-small cell lung cancer (NSCLC). Through inhibition of EGFR phosphorylation, the drug inhibits the downstream signal transduction and cell proliferation. In the in vivo tumor xenograft model of NSCLC and head and neck squamous cell carcinoma, erlotinib demonstrates anticancer effect by inhibiting tumor cell growth or inducing apoptosis of tumor cells. The experiment showed that erlotinib oral bioavailability is 80%. (1 TSAO, M S, Erlotinib in lung Cancer-Molecular and clinical predictors of outcome, New England Journal of Medicine, 2005, 353:133-144; 2 WONG M, K, LO A I, LAM B, et al. Erlotinib as maintenance treatment after failure to first-line gefitinib in non-small cell lung cancer. Cancer Chemother Pharmacol, 2010, 65 (6): 1023-1028).
(3) Lapatinib (Lapatinib/Tykerb/GW572016). Lapatinib is EGFR/HER2 quinazoline inhibitors with dual target, developed by GlaxoSmithKline, which was approved in the United States in March 2007. Lapatinib can suppress both the EGFR and HER2 tyrosine kinase and its downstream MARK and PI3K signal transduction, thereby blocking the proliferation of cancer cells. Lapatinib is used for the treatment of non-small cell lung cancer and breast cancer (1, WOOD, E R, TRUESDALE A T, MCDONALD O B, et al, A unique structure for epidermal growth factor receptor bound to GW572016 (Lapatinib): relationships among protein conformation, inhibitor off-rate, and receptor activity in tumor cells, Cancer Research 2004, 64:6652-659; 2, GEYER C, FORSTER J, LINDQUIST D, et al, Lapatinib plus capecitabine for HER2 positive advanced breast cancer, New England Journal of Medicine, 2006, 355 (26):2733-2743).
(4) Icotinib (Icotinib, Conmana). Icotinib was approved in June 2011 by CFDA, this is China's first small molecules anti-tumor drug with independent intellectual property rights for the treatment of advanced non-small cell lung cancer. Icotinib retained core structure of quinazoline, the only difference is in the side chain with closed loop. The biological testing at molecular level showed that icotinib IC50 was 5 nmol/mL, which show strong inhibitory effect against EGFR activity. From the screening of 85 kinases, icotinib selectively inhibits EGFR and 3 other mutants. It had no significant effect on the remaining 81 kinase. Icotinib showed good safety and tolerability profile (SHAO J H, GUO J X, ZHANG X D, et al. Synthesis and biological evaluation of crown ether fused quinazoline analogues as potent EGFR inhibitors, Bioorganic & Medicinal Chemistry Letters, 2012, 12:6301-6305).
EGFR inhibitors have achieved a certain effect on the treatment to advanced non-small cell lung cancer, but the resistance phenomenon has appeared in these drugs (Qi Li, Yali Zhao, Xianghong Li, Study on EGFR Gene Mutation in non-small Cell Lung Cancer, Chinese Journal of Oncology, 2007, 29 (4): 270). Because EGFR signaling pathways are involved in a variety of functions of the mediate cells, their drug resistance may be associated with the disorder of multiple signal conducting pathways, including drug resistance mutations, structural activation, and bypass activation of downstream signal. Common resistance mechanisms are as follows: 1) T790M mutation. The study found that EGFR mutations are found in many patients with EGFR-TKI resistance. T790M mutation (the tyrosine kinase active site 790 threonine mutates to methionine) was first proposed by Kobayashi in 2005 (KOBAYASHI S, BOGGON T J, DAYARAM T, et al. EGFR mutation and resistance of non-small cell lung cancer to gefitinib, New England Journal of Medicine, 2005, 352(8): 786-792) and is the most common secondary mutation. EGFR T790M mutations are found in approximately 50% of the drug-resistant patients. Through the EGFR gene detection of tumor tissue, it is found that No. 20 exon of EGFR had a secondary mutation, leading to tyrosine kinase 790 threonine is replaced by methionine, resulting in the emergence of drug resistance. The resistance caused by the T790M mutation is confirmed in subsequent studies. This may be due to the fact that the mutations increase the affinity of the active site of EGFR with the ATP and the binding of TKIs to EGFR is hindered. (ENGELMAN J A, ZEJNULLAHU K, MITSUDOMI T, et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 Signaling, Science, 2007, 316(5827): 1039-1043) 2) C-MET amplification. C-MET is a proto-oncogene whose coded protein is a receptor for hepatocyte growth factor (HGF) with tyrosine kinase activity. Engelman et al. proposed that C-MET amplification is another major mechanism for EGFR-TKIs-acquired drug resistant mutations, which accounts for 20% of all drug resistance. The amplification of C-MET activates the ErbB3/PI3K/AKT signaling pathway, leading to resistance of NSCLC patients to EGFR-TKIs.
It has been found that EGFR and its family receptor are overexpressed in tumor cells of incident cancer such as lung cancer. The mechanism of molecular biology indicates that abnormal expression of EGFR and its signal transduction have an important effect on the proliferation of tumor cells. Some EGFR-targeted small molecule inhibitors have been developed and used in the treatment of advanced non-small cell lung cancer. However, due to occurrence of the drug resistance mutations, the survival time of most advanced patients still needs to be improved.